Isolation and physiological properties of methanogenic archaea that degrade tetramethylammonium hydroxide.

Tetramethylammonium hydroxide (TMAH) is a known toxic chemical used in the photolithography process of semiconductor photoelectronic processes. Significant amounts of wastewater containing TMAH are discharged from electronic industries. It is therefore attractive to apply anaerobic treatment to industrial wastewater containing TMAH. In this study, a novel TMAH-degrading methanogenic archaeon was isolated from the granular sludge of a psychrophilic upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater containing TMAH. Although the isolate (strain NY-STAYD) was phylogenetically related to Methanomethylovorans uponensis, it was the only isolated Methanomethylovorans strain capable of TMAH degradation. Strain NY-STAYD was capable of degrading methylamine compounds, similar to the previously isolated Methanomethylovorans spp. While the strain was able to grow at temperatures ranging from 15 to 37°C, the cell yield was higher at lower temperatures. The distribution of archaeal cells affiliated with the genus Methanomethylovorans in the original granular sludge was investigated by fluorescence in situ hybridization (FISH) using specific oligonucleotide probe targeting 16S rRNA. The results demonstrated that the TMAH-degrading cells associated with the genus Methanomethylovorans were not intermingled with other microorganisms but rather isolated on the granule's surface as a lone dominant archaeon. KEY POINTS: • A TMAH-degrading methanogenic Methanomethylovorans strain was isolated • This strain was the only known Methanomethylovorans isolate that can degrade TMAH • The highest cell yield of the isolate was obtained at psychrophilic conditions.

Use of Pressure Activation in Food Quality Improvement.

Beside intensive studies on inactivation microorganisms by high hydrostatic pressure (HP) for food storage, pressure effects on property of food materials have also been studied based on knowledge in pressure effect on biomolecules. Pressure effects on biological membranes and mass transfer in cellular biological materials and on enzyme activity would give an idea that HP treatment can introduce two types of activations into food materials: improved mass transfer and enzyme activity. Studies focusing on these pressure activations on food materials were then reviewed. Rice flour with an exclusively fine mean particle size and small starch damage was obtained due to improved water absorption properties and/or enzyme activity by HP. HP treatment increased of free amino acids and γ-aminobutyric acid (GABA) in rice and soybeans due to improved proteolysis and amino acid metabolism. Improvement of antioxidant activity and alteration of polyphenolic-compounds composition in food materials were also demonstrated by HP treatment. The HP-induced activations on food materials could contribute towards processing technologies for food quality improvement.

Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters.

NhaB-like antiporters were the second described class of Na(+)/H(+) antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent Km values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent Km at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of Km in the course of molecular evolution.

Enzymatic production of γ-aminobutyric acid in soybeans using high hydrostatic pressure and precursor feeding.

The effects were investigated of the glutamic acid (Glu) substrate concentration on the generation and kinetics of γ-aminobutyric acid (GABA) in soybeans treated under high hydrostatic pressure (HHP; 200 MPa for 10 min at 25 °C). The conversion of Glu to GABA decreased with increasing initial Glu concentration in the soybeans. The crude glutamate decarboxylase (GAD) obtained from the HHP-treated soybeans showed substrate inhibition. The GABA production rate in the HHP-treated soybeans fitted the following substrate inhibition kinetic equation: v0=(VmaxS0)/(Km+S0+(S0)2/Ki). The Km value for the HHP-treated soybeans was significantly higher than that of the untreated soybeans. The Km values in this study show the affinity between Glu and GAD, and indicate that the HHP-treated soybeans had lower affinity between Glu and GAD than the untreated soybeans. GAD extracted from the HHP-treated soybeans showed a similar value to that in the HHP-treated soybeans. The intact biochemical system was so damaged in the HHP-treated soybeans that it showed substrate inhibition kinetics similar to that of the extracted GAD. The combination of HHP and precursor feeding proved to be a novel tool that can be used to increase the concentration of a target component.

Detection of active, potentially acetate-oxidizing syntrophs in an anaerobic digester by flux measurement and formyltetrahydrofolate synthetase (FTHFS) expression profiling.

Syntrophic oxidation of acetate, so-called reversed reductive acetogenesis, is one of the most important degradation steps in anaerobic digesters. However, little is known about the genetic diversity of the micro-organisms involved. Here we investigated the activity and composition of potentially acetate-oxidizing syntrophs using a combinatorial approach of flux measurement and transcriptional profiling of the formyltetrahydrofolate synthetase (FTHFS) gene, an ecological biomarker for reductive acetogenesis. During the operation of a thermophilic anaerobic digester, volatile fatty acids were mostly depleted, suggesting a high turnover rate for dissolved H(2), and hydrogenotrophic methanogens were the dominant archaeal members. Batch cultivation of the digester microbiota with (13)C-labelled acetate indicated that syntrophic oxidation accounted for 13.1-21.3 % of methane production from acetate. FTHFS genes were transcribed in the absence of carbon monoxide, methoxylated compounds and inorganic electron acceptors other than CO(2), which is implicated in the activity of reversed reductive acetogenesis; however, expression itself does not distinguish whether biosynthesis or biodegradation is functioning. The mRNA- and DNA-based terminal RFLP and clone library analyses indicated that, out of nine FTHFS phylotypes detected, the FTHFS genes from the novel phylotypes I-IV in addition to the known syntroph Thermacetogenium phaeum (i.e. phylotype V) were specifically expressed. These transcripts arose from phylogenetically presumed homoacetogens. The results of this study demonstrate that hitherto unidentified phylotypes of homoacetogens are responsible for syntrophic acetate oxidation in an anaerobic digester.

Genomic and Metabolomic Analyses of a Piezosensitive Mutant of Saccharomyces cerevisiae and Application for Generation of Piezosensitive Niigata-Sake Yeast Strains.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Formation of green-blue compounds in Brassica rapa root by high pressure processing and subsequent storage.

The effect of high pressure treatment on biochemical changes during storage was investigated using Brassica rapa root. High pressure treated samples with 400 and 600 MPa formed unique green-blue color during 7-d storage at 4 degrees C. The mechanism of green-blue compound formation would be based on biochemical pathway for a unique green-blue pigment synthesis, containing O2-dependent steps and possibly enzymatic reactions.

Immunostimulation-mediated antitumor activity by preconditioning with rice-shochu distillation residue against implanted tumor in mice.

We investigated the ability of rice-shochu postdistillation residue (RSDR) to stimulate the activity of macrophages. RSDR significantly stimulated mouse macrophage activity and induced significant IL-12 production in vitro. In syngeneic C38 solid tumor model in mice, a diet containing 1.0% RSDR caused a significant suppression of tumor growth and prolonged the life span of the tumor-bearing mice. Further, using this model, mice fed for 21 days with RSDR showed significantly increased levels of serum IL-12 and IFN-gamma compared with controls. Moreover, the splenic NK cell activity of mice fed with RSDR was significantly elevated compared with that of mice on a normal diet and thereby suppressed C38 tumor growth. We also investigated the tumor growth suppressing effect of RSDR using a tumor model of B16-F10 melanoma cells. Dietary preconditioning with RSDR significantly suppressed B16-F10 tumor growth. Moreover, RSDR significantly increased the production of IL-12 either before or after B16-F10 tumor implantation. These results suggest that dietary RSDR suppresses tumor growth by stimulating the immune system of the host.

Antioxidant activity of vinegar produced from distilled residues of the Japanese liquor shochu.

Rice shochu distilled residue (RSDR) is a byproduct of rice shochu production. RSDR was converted into vinegar by acetate fermentation. In our present study, two major antioxidant compounds, tyrosol and ferulic acid, were identified from the RSDR-derived vinegar. Furthermore, we investigated the antioxidant activity of freeze-dried RSDR-derived vinegar, which was Acetobactor aceti fermentation powder (AFP), in vitro and in vivo. AFP at 0.25 mg/mL or higher concentrations showed an inhibitory effect against lipid peroxidation and cellular GSH depletion in HepG2 cells induced by H(2)O(2) (P < 0.05). We thus considered the potential of AFP in protecting cells against damage induced by H(2)O(2). Its antioxidant activity was evaluated in vivo using carbon tetrachloride (CCl(4))-induced acute liver injury mouse models. Five consecutive days of oral preadministration of AFP dissolved in PBS at 200, 400, and 800 mg/kg body weight significantly suppressed lipid peroxidation in the liver induced by CCl(4) (P < 0.01). Consequently, treatment with AFP at 200 mg/kg body weight or higher doses suppressed the elevation of alanine aminotransferase and aspartate aminotransferase levels in serum (P < 0.05). These findings suggest that RSDR-derived vinegar can be developed as a health food with an antioxidant effect for the prevention of oxidative injury and cancer.

Comparative analyses of viable bacterial counts in foods and seawater under microplate based liquid- and conventional agar plate cultivation: increased culturability of marine bacteria under liquid cultivation.

Bacterial counts under liquid cultivation using 96-well microplates were performed. The counts under liquid and under solid cultivation were equivalent in foods, although the counts under liquid cultivation exceeded those under solid cultivation in seawater, suggesting that some bacteria in seawater were viable but did not form detectable colonies. Phylogenetic analysis of bacteria obtained under liquid cultivation was also performed.

Anaerobic treatment performance and microbial population of thermophilic upflow anaerobic filter reactor treating awamori distillery wastewater.

Distillery wastewater from awamori making was anaerobically treated for one year using thermophilic upflow anaerobic filter (UAF) reactors packed with pyridinium group-containing nonwoven fabric material. The microbial structure and spatial distribution of microorganisms on the support material were characterized using molecular biological methods. The reactor steadily achieved a high TOC loading rate of 18 g/l/d with approximately 80% TOC removal efficiency when non-diluted wastewater was fed. The maximum TOC loading rate increased to 36 g/l/d when treating thrice-diluted wastewater. However, the TOC removal efficiency and gas evolution rate decreased compared with that when non-diluted wastewater was used. Methanogens closely related to Methanosarcina thermophila and Methanoculleus bourgensis and bacteria in the phyla Firmicutes and Bacteroidetes were predominant methanogens and bacteria in the thermophilic UFA reactor, as indicated by 16S rRNA gene clone analysis. Fluorescence in situ hybridization (FISH) results showed that a large quantity of bacterial cells adhered throughout the whole support, and Methanosarcina-like methanogens existed mainly in the relative outside region while Methanoculleus cells were located in the relative inner part of the support. The support material used proved to be an excellent carrier for microorganisms, and a UAF reactor using this kind of support can be used for high-rate treatment of awamori/shochu distillery wastewater.

Comparative analysis on inactivation kinetics of between piezotolerant and piezosensitive mutant strains of Saccharomyces cerevisiae under combinations of high hydrostatic pressure and temperature.

We previously obtained a pressure-tolerant (piezotolerant) and a pressure sensitive (piezosensitive) mutant strain, under ambient temperature, from Saccharomyces cerevisiae strain KA31a. The inactivation kinetics of these mutants were analyzed at 150 to 250MPa with 4 to 40°C. By a multiple regression analysis, the pressure and temperature dependency of the inactivation rate constants k values of both mutants, as well as the parent strain KA31a, were well approximated with high correlation coefficients (0.92 to 0.95). For both mutants, as well as strain KA31a, the lowest k value was shown at a low pressure levels with around ambient temperature. The k value approximately increased with increase in pressure level, and with increase and decrease in temperature. The piezosensitive mutant strain a924E1 showed piezosensitivity at all pressure and temperature levels, compared with the parent strain KA31a. In contrast, the piezotolerant mutant strain a2568D8 showed piezotolerance at 4 to 20°C, but did not show significant piezotolerance at 40°C. These results of the variable influence of temperature on pressure inactivation of these strains would be important for better understanding of piezosensitive and piezotolerant mechanisms, as well as the pressure inactivation mechanism of S. cerevisiae.

Effect of high pressure on the saccharification of starch in the tuberous root of sweet potato (Ipomoea batatas).

We analyzed the effect of high hydrostatic pressure (HHP) treatment on reducing sugar production in the tuberous root of sweet potato (Ipomoea batatas), based on pressure-gelatinization of starch and subsequent saccharification by internal amylases. HHP treatment at up to 600MPa at ambient temperature for 10min did not apparently affect the reducing sugar concentration in tuberous root. However, HHP treatment at 100 to 500MPa and 60°C or 70°C for 10min increased reducing sugar concentration as both the pressure and temperature increased. The reducing sugar concentration after HHP treatment at 500MPa and 70°C for 10min was roughly comparable to that of the thermal treatment control (80°C for 10min under atmospheric pressure). HHP treatment enabled the gelatinization and enzymatic saccharification of starch in the tuberous root of sweet potato, at a lower temperature than required by thermal treatment at atmospheric pressure.

Microbial diversity of mesophilic methanogenic consortium that can degrade long-chain fatty acids in chemostat cultivation.

We established a chemostat cultivation method for a mesophilic methanogenic consortium that could degrade long-chain fatty acids (LCFA) using a completely stirred tank reactor (CSTR) fed with synthetic wastewater containing oleic and palmitic acids as the carbon and energy sources. The critical dilution rate of the chemostat, in which most of the introduced LCFA were decomposed and mineralized, was 0.4 d(-1). The microbial community under steady-state condition at this dilution rate was analyzed by 16S rRNA gene sequencing. We detected the following major groups of methanogens within the archaeal community: the aceticlastic genera Methanosaeta and Methanosarcina and the hydrogenotrophic genus Methanospirillum. We also detected organisms that were closely related to fatty-acid oxidizing bacteria affiliated with the family Syntrophomonadaceae. However, bacteria belonging to the phyla Bacteroidetes and Spirochaetes, which are phylogenetically distant from known fatty-acid oxidizing bacteria, apparently predominated in the population, indicating that they play important roles in LCFA degradation within the chemostat.

Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation.

Two mesophilic anaerobic chemostats, one without added Ni2+ and Co2+ (chemostat 1) and the other with added Ni2+ and Co2+ (chemostat 2), were supplied with synthetic wastewater containing bovine serum albumin (BSA) as the sole carbon and energy source in order to study the capacity of protein degradation, microbial community structure and the effects of the addition of trace metals. Volatile fatty acids and ammonia were the main products of chemostat 1, while methane, CO2 and ammonia were the main products of chemostat 2, and critical dilution rates of 0.15 d-1 and 0.08 d-1 were obtained, respectively. Fluorescence in situ hybridization (FISH) with archaeal and bacterial domain-specific probes showed that archaeal cells were very limited in chemostat 1 while large populations of several types of archaeal cells were present in chemostat 2. Phylogenetic analyses based on 16S rRNA gene clonal sequences, DGGE, and quantitative real-time polymerase chain reaction (PCR) showed that, within the domain Archaea, methanogens affiliated with the genera Methanosaeta and Methanoculleus were predominant in chemostat 2. Within the domain Bacteria, rRNA genes obtained from chemostat 1 were affiliated with the three phyla; Firmicutes (43%), Bacteroidetes (50%) and Proteobacteria (7%). A total of 56% of rRNA genes obtained from chemostat 2 was affiliated with the three phyla, Firmicutes (32%), Bacteroidetes (11%) and Proteobacteria (13%) while 44% of rRNA genes remained unclassified. Phylogenetically distinct clones were obtained in these two chemostats, suggesting that different protein degradation pathways were dominant in the two chemostats: coupled degradation of amino acids via the Stickland reaction in chemostat 1 and uncoupled degradation of amino acids via syntrophic association of amino acid degraders and hydrogenotrophic methanogens in chemostat 2.

Cellulose production from glucose using a glucose dehydrogenase gene (gdh)-deficient mutant of Gluconacetobacter xylinus and its use for bioconversion of sweet potato pulp.

A gene fragment encoding a putative pyrroloquinoline quinone glucose dehydrogenase (PQQ GDH) was cloned from a bacterial cellulose (BC)-forming acetic acid bacterium, Gluconacetobacter xylinus (=Acetobacter xylinum) strain BPR 2001, which was isolated as a high BC producer when using fructose as the carbon source. A GDH-deficient mutant of strain BPR 2001, namely GD-I, was then generated via gene disruption using the cloned gene fragment. Strain GD-I produced no gluconic acid but produced 4.1 g.l(-1) of BC aerobically in medium containing glucose as the carbon source. The ability of strain GD-I to convert glucose to BC was approximately 1.7-fold higher than that of the wild type. Strain GD-I was also able to produce 5.0 g.l(-1) of BC from a saccharified solution, which was derived from sweet potato pulp by enzymatic saccharification. Supplementation of ethanol during aerobic cultivation further increased the concentration of BC produced by strain GD-I to 7.0 g.l(-1). The rate of conversion from glucose to BC under these cultivation conditions was equivalent to that of strain BPR 2001 cultivated with fructose as the carbon source.

Barosensitivity in Saccharomyces cerevisiae is Closely Associated with a Deletion of the COX1 Gene.

High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high-value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high-pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high-pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure-sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria.

Effects of High Hydrostatic Pressure on Water Absorption of Adzuki Beans.

The effect of high hydrostatic pressure (HHP) treatment on dried soybean, adzuki bean, and kintoki kidney bean, which are low-moisture-content cellular biological materials, was investigated from the viewpoint of water absorption. The samples were vacuum-packed with distilled water and pressurized at 200 MPa and 25 °C for 10 min. After the HHP treatment, time courses of the moisture contents of the samples were measured, and the dimensionless moisture contents were estimated. Water absorption in the case of soybean could be fitted well by a simple water diffusion model. High pressures were found to have negligible effects on water absorption into the cotyledon of soybean and kintoki kidney bean. A non-linear least square method based on the Weibull equation was applied for the adzuki beans, and the effective water diffusion coefficient was found to increase significantly from 8.6 × 10-13 to 6.7 × 10-10 m²/s after HHP treatment. Approximately 30% of the testa of the adzuki bean was damaged upon HHP treatment, which was comparable to the surface area of the testa in the partially peeled adzuki bean sample. Thus, HHP was confirmed to promote mass transfer to the cotyledon of legumes with a tight testa.

Dynamics of the microbial community during continuous methane fermentation in continuously stirred tank reactors.

Methane fermentation is an attractive technology for the treatment of organic wastes and wastewaters. However, the process is difficult to control, and treatment rates and digestion efficiency require further optimization. Understanding the microbiology mechanisms of methane fermentation is of fundamental importance to improving this process. In this review, we summarize the dynamics of microbial communities in methane fermentation chemostats that are operated using completely stirred tank reactors (CSTRs). Each chemostat was supplied with one substrate as the sole carbon source. The substrates include acetate, propionate, butyrate, long-chain fatty acids, glycerol, protein, glucose, and starch. These carbon sources are general substrates and intermediates of methane fermentation. The factors that affect the structure of the microbial community are discussed. The carbon source, the final product, and the operation conditions appear to be the main factors that affect methane fermentation and determine the structure of the microbial community. Understanding the structure of the microbial community during methane fermentation will guide the design and operation of practical wastewater treatments.